The proposed research examines Moloney murine leukemia virus as a model system for testing determinants and outcomes of general and specific features of retroviral genome structure. One hypothesis is that previously unrecognized genome features contribute to specific replication steps such as template recognition, primer generation and primer utilization. Another hypothesis is that retroviruses can accommodate significant deviation from their usual genome form, and that even rare or transient genomic deviation can fuel the evolution of retroviral populations. Subsequent hypotheses are more specific corollaries of the above, such as that dimerization does not occur randomly among packaging-competent RNAs, and that general genome factors such as RNA length can affect the fidelity of genome replication due to virus assembly defects. Most of the proposed experiments characterize the replication properties of retroviral vectors engineered to serve as assays during replication in cultured cells. The first specific aim tests size limitations imposed on retroviral genomes and examines the consequences and implications of these limitations during several stages of the replication cycle. The second aim addresses, which MLV RNAs are capable of joining in a dimer, and other determinants of virion RNA genome organization and composition. The third aim examines two different viral genome 3' end features and the ways in which interactions between these features and viral or host factors influence the form and functioning of retroviral genomes. The long-term objectives of the proposed research are to better understand normal MLV genome metabolism as well as to understand how these compare to properties of other retroviral species. This work will also reveal what deviation can occur and can contribute to the virus' genomic repertoire. Structural variation is important to the pathogenesis of retroviruses. For example, aberrant Moloney murine leukemia virus genomes frequently accumulate during the progression to cancer in mice, and genomic variation is also important in determining the pathogenic properties of human disease-causing retroviruses such as HIV- I. Examining limitations imposed by specific genome features and understanding parameters that affect specific steps in the viral genome replication cycle will also assist the development of improved retroviral gene delivery vectors.